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Interactions of eosin with three different substrates, β-lactoglobuline, bovine serum albumin and cysteine, in aqueous solutions of pH 7 under illumination with light of wavelengths 5200—5400 Å are investigated by changes in absorption spectrum characteristics, SH-group activities and phosphorescence intensities.
Only with bovine serum albumin the major part of protein conversion, as shown by spectral changes and diminution of SH-groups due to eosin-sensitized photo-oxidation. In β-lactoglobuline an oxidizing photoreaction occurs, by which eosin is vanishing to the same degree as the protein shows loss of SH-groups and spectral alterations indicating attack on aromatic amino acid residues. There is no red shift of the eosin absorption band at 5170 Å as is observed in solutions of bovine serum albumin, where the intensity of phosphorscence is about 100 fold compared with the intensity obtained by solutions of β-lactoglobulin.
The aerobic eosin photoreaction in solutions of β-lactoglobulin is faster than aerobic photobleaching of the dye. Still faster is its bleaching photoreaction with cysteine, which is nearly independent of oxygen.
In order to determine the influence of OH and O2H-radicals on proteins, bovine serum albumin (BSA) in aqueous solution was treated with Fenton’s reagent [Fe(II)SO4+EDTA+H2O2] and with ultraviolet light (λ > 2800 Å) in the presence of H2O2. The action of free radicals produced in this way did not change the properties of the native protein with respect to the sedimentation in the ultracentrifuge or optical rotatory dispersion and electrophoresis under normal conditions. Ampèrometric titration indicated partial oxidation of SH-groups and of 3—5 SS-groups which are not reducible by NaBH4.
Heat aggregation investigated by means of light-scattering was suppressed at pH 7.5 and strongly accelerated at pH 4.6 (range of coagulation), the latter being a result of increased entropy of activation of coagulation velocity.
The difference spectrum against native BSA had positive values of Δε and two maxima at 2480 and 2950 Å.
Ultracentrifugation at room temperature in phosphate buffer (pH 7.3, μ=0.18) furnishes a molecular weight of 63 300. In a solution of 8 M urea and borate buffer (pH 9, μ=0.05) fragments with molecular weights between 25 000 and 37 000 were observed while in phosphate buffer (pH 7.3, without urea) at temperatures higher than 46 °C an anomalous behaviour of the concentration gradient indicated an effect which possibly depends on a dissociation equilibrium.
As a consequence oxygen radicals seem to attack not only SH- and SS-groups but at least one covalent bond of the peptide chain. Some experiments of heat aggregation with BSA treated with γ-rays (60Co) gave the same results as BSA treated with Fenton’s reagent or UV-light+H2O2.
The thermal decomposition of 1,2-diadamantyldioxetane was studied by kinetic and spectroscopic methods. Spectra of the chemiluminescence emitted during the thermally induced decomposition of 1,2-diadamantyldioxetane, tetramethyldioxetane and trimethyldioxetane were obtained and the influence of quenchers and radical-scavengers, and the presence of "heavy atoms" in the surrounding of the emitting species was investigated. The kinetics of the decay mechanism was followed by measuring the time dependence of the chemiluminescence. The influence of radical-scavengers, quenchers and "external heavy atoms" on the kinetics was assessed. Experimental results were discussed in terms of a biradical decay mechanism.
Diadamantyldioxetane, trim ethyldioxetane and tetram ethyldioxetane were photolyzed b y light of A > 260 nm . The spectral distribution o f the quanta emitted during photoinduced decom position of dioxatenes was found to be different from fluorescence and phosphorescence o f ketones. Flash photolysis experim ents showed the absorption of an short-lived interm ediate. It was concluded, therefore, that photolysis o fdioxetanes is not a concerted process but involves at least one precursor o f the final product ketone.
The carcinogenic hydrocarbon 3.4-benzopyrene is soluble in aqueous solutions of different proteins. The solubilities are easily determined by the fluorimetric method. The fluorescence o. the hydrocarbon in the protein solutions is not quenched by molecular oxygen. Nevertheless only in presence of air (oxygen) an irreversible decrease of the fluorescence intensity occurs under irradiation with UV-light of wavelength 366 mμ, which is considerably faster than under nitrogen or in solutions of the hydrocarbon in ethanol or aqueous caffeine.
In the systems investigad, a correlation was found between the half-life period of the reaction and the SH-group activities. The participation of protein-SH-Groups in the 3.4-benzopyrene photoreaction is demonstrated by ampèrometric Ag⊕-titrations.
The influence of protein denaturation and inhibiting additives on the photoreaction are investigated by the fluorimetric method.
Irradiation- and oxygen-dependence of the reaction are analogous to the observations of photodynamic action and skin cancer induction by 3.4-benzopyrene.
By 366 mµ irradiation of β-lactoglobuline solutions containing 3.4-benzopyrene the heatdenaturation characteristics of the protein are changed. The same changes are produced without 3.4-benzopyrene by UV-light of the wavelength 280 mµ. Treatment of the β-lactoglobuline solutions with an amount of cigarette smoke, which certainly does not contain 3.4-benzopyrene in sufficient concentration, acts in the same direction.
Along with the changes in the protein properties the typical fluorescence of 3.4-benzopyrene vanishes. The hydrocarbon does not act as a catalyst in photodynamic action, but is chemically altered as well as the protein, at least in the system under investigation.
Durch fluoreszenz-spektrographische Intensitätsmessungen mit einem aus Laboratoriumsmitteln gebauten, einfachen Fluoreszenzspektrometer wurde die Löslichkeit des carcinogenen Kohlenwasserstoffs 3.4-Benzpyren in verdünnten, wäßrigen Lösungen von β-Lactoglobulin und Milchsäure-Dehydro-genase bestimmt. Die molare Lösungsvermittlung der Proteine für 3.4-Benzpyren ist erheblich größer als die des Koffeins. Die Fluoreszenz des 3.4-Benzpyrens wird in den Proteinlösungen nicht durch molekularen Sauerstoff gelöscht. Das beobachtete Spektrum gleicht demjenigen alkoholischer Lösungen von 3.4-Benzpyren ohne Konzentrationslöschung.
Lumineszenz von Hefe
(1968)